Far-field optical super-resolution microscopy techniques may aim to resolve features in a specimen substantially smaller than the classical Abbe diffraction resolution limit. Typically, the specimen may be biological and the features of interest may be labeled with fluorescent molecules or quantum dots. Some super-resolution techniques such as Structured Illumination Microscopy (SIM) (see for example, Gustafsson, M. G. L., J. Microsc., vol. 198, pt. 2, 2000, incorporated herein by reference) may illuminate the specimen sequentially with phase-shifted periodically-structured light, sometimes formed by an image of a shifted grating, to convert spatial frequencies that are classically unresolvable to resolvable spatial frequencies through a process analogous to the Moiré effect, thereby surpassing the Abbe limit by up to a factor of 2 in an epi-fluorescence microscope, or in some cases more if the fluorescence response to the excitation is nonlinear.
Lateral modulation of the specimen with phase-shifted incoherent structured illumination may also be utilized to reject background light and improve optical sectioning in a wide-field microscope (see, for example, Neil, M. A. A., Juskaitis, R. and Wilson, T., Optics Letters, vol. 22, 1997, incorporated herein by reference), whereas axially-varying structured illumination generated at the specimen by opposing microscope objectives may be used to improve depth resolution beyond the classical limit imposed by the diffraction-limited depth of field. For example, in Spatially Modulated Illumination (SMI) microscopy, disclosed in U.S. Pat. No. 7,298,461, incorporated herein by reference, a sub-wavelength sized fluorescent object moved axially through an apodized structured illumination field between two opposing objectives may produce a modulated detector signal, which may be fitted to a function to estimate the emitter's size and distance with respect to other emitters in the field with a resolution exceeding the classical limit.
Other super-resolution techniques, such as Stimulated Emission Depletion (STED) microscopy (see Hell, S. W., Wichmann, J., Optics Letters, vol. 19, no. 11, 1994, incorporated herein by reference) may improve resolution further by exploiting a non-linear fluorescence excitation process. In STED, for example, the specimen may be scanned with a doughnut-shaped focal spot temporarily depleting fluorescence of specialized fluorophores outside a small central spot, which may be much smaller than the Abbe limit. The undepleted fluorophores within the spot may then be measured and localized using a conventional focused excitation beam at a different wavelength.
Other super-resolution approaches may exploit stochastic properties of fluorescence to isolate and localize individual fluorophores with precision exceeding the Abbe limit. For example, Photoactivated Localization Microscopy (PALM) (see Betzig, E. et al., Science, vol. 313, no. 5793, 2006, incorporated herein by reference) and Stochastic Optical Reconstruction Microscopy (STORM) (see Rust. NI, J., Bates, M., Zhuang, X., Nature Methods, vol. 3, no. 20, 2006, incorporated herein by reference) employ photoswitchable fluorescent molecules, the majority of which are in a dark state at any given time and do not fluoresce in response to an excitation laser. However, a weak activation laser at another wavelength (or in some cases the same excitation laser) may be used to temporarily switch a small fraction of the molecules into an active state stochastically, resulting in a sparse distribution at any given time of mutually-isolated molecules which may fluoresce in response to the excitation laser. An image of the sparsely-distributed fluorescing molecules formed on a camera may be used to find their locations by estimating the centers of the respective point spread functions (PSFs) with a precision which may be substantially finer than the size of the PSF or the size of a pixel. After a short time, an active molecule may re-enter the dark state, may photobleach, or may be deactivated with another laser, while a new molecule may become active.
Localizations of different sparse sets of active molecules using a sequence of images may be combined to form a super-resolved image of the specimen if the density of fluorophores is sufficient to adequately sample the labeled features of interest. Localization precision may be limited by various factors such as the number of photons emitted from each molecule, background fluorescence, and the number of pixels sampling the PSF. In some cases, nine or more pixels may be needed to sufficiently localize a focused PSF distribution at the image plane, which may limit the measurement field of view, and/or acquisition speed. Furthermore, some activated molecules may reside outside the depth of field of the imaging system and their images may appear out of focus, requiring even more pixels for localization, limiting localization precision, and contributing background noise to in-focus emitter images. Auto-fluorescence from the specimen volume and residual fluorescence from de-activated molecules may compound background noise and further degrade attainable resolution.